Combining metabolic engineering and biocompatible chemistry for high-yield production of homo-diacetyl and homo-(S,S)-2,3-butanediol

Biocompatible chemistry is gaining increasing attention because of its potential within biotechnology for expanding the repertoire of biological transformations carried out by enzymes. Here we demonstrate how biocompatible chemistry can be used for synthesizing valuable compounds as well as for linking metabolic pathways to achieve redox balance and rescued growth. By comprehensive rerouting of metabolism, activation of respiration, and finally metal ion catalysis, we successfully managed to convert the homolactic bacterium Lactococcus lactis into a homo-diacetyl producer with high titer (95 mM or 8.2 g/L) and high yield (87% of the theoretical maximum). Subsequently, the pathway was extended to (S,S)-2,3-butanediol (S-BDO) through efficiently linking two metabolic pathways via chemical catalysis. This resulted in efficient homo-S-BDO production with a titer of 74 mM (6.7 g/L) S-BDO and a yield of 82%. The diacetyl and S-BDO production rates and yields obtained are the highest ever reported, demonstrating the promising combination of metabolic engineering and biocompatible chemistry as well as the great potential of L. lactis as a new production platform.     

Biotechnological production of γ-decalactone,a peach like aroma,by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

The request for new flavourings increases every year. Consumer perception that everything natural is better is causing an increase demand for natural aroma additives. Biotechnology has become a way to get natural products. γ-Decalactone is a peach-like aroma widely used in dairy products, beverages and others food industries. In more recent years, more and more studies and industrial processes were endorsed to cost-effect this compound production. One of the best-known methods to produce γ-decalactone is from ricinoleic acid catalyzed by Yarrowia lipolytica, a generally regarded as safe status yeast. As yet, several factors affecting γ-decalactone production remain to be fully understood and optimized. In this review, we focus on the aromatic compound γ-decalactone and its production by Y. lipolytica. The metabolic pathway of lactone production and degradation are addressed. Critical analysis of novel strategies of bioprocess engineering, metabolic and genetic engineering and other strategies for the enhancement of the aroma productivity are presented.     

Medicinally important secondary metabolites in recombinant microorganisms or plants: Progress in alkaloid biosynthesis

Plants produce a high diversity of natural products or secondary metabolites which are important for the communication of plants with other organisms. A prominent function is the protection against herbivores and/or microbial pathogens. Some natural products are also involved in defence against abiotic stress, e.g. UV-B exposure. Many of the secondary metabolites have interesting biological properties and quite a number are of medicinal importance. Because the production of the valuable natural products, such as the anticancer drugs paclitaxel, vinblastine or camptothecin in plants is a costly process, biotechnological alternatives to produce these alkaloids more economically become increasingly important. This review provides an overview of the state of art to produce alkaloids in recombinant microorganisms, such as bacteria or yeast. Some progress has been made in metabolic engineering usually employing a single recombinant alkaloid gene. More importantly, for benzylisoquinoline, monoterpene indole and diterpene alkaloids (taxanes) as well as some terpenoids and phenolics the proof of concept for production of complex alkaloids in recombinant Escherichia coli and yeast has already been achieved. In a long-term perspective, it will probably be possible to generate gene cassettes for complete pathways, which could then be used for production of valuable natural products in bioreactors or for metabolic engineering of crop plants. This will improve their resistance against herbivores and/or microbial pathogens.     

Artificially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies

Starch/cellulose has become the major feedstock for manufacturing biofuels and biochemicals because of their abundance and sustainability. In this study, we presented an artificially designed “starch-mannose-fermentation” biotransformation process through coupling the advantages of in vivo and in vitro metabolic engineering strategies together. Starch was initially converted into mannose via an in vitro metabolic engineering biosystem, and then mannose was fermented by engineered microorganisms for biomanufacturing valuable mannosyl compounds. The in vitro metabolic engineering biosystem based on phosphorylation/dephosphorylation reactions was thermodynamically favorable and the conversion rate reached 81%. The mannose production using whole-cell biocatalysts reached 75.4 g/L in a 30-L reactor, indicating the potential industrial application. Furthermore, the produced mannose in the reactor was directly served as feedstock for the fermentation process to bottom-up produced 19.2 g/L mannosyl-oligosaccharides (MOS) and 7.2 g/L mannosylglycerate (MG) using recombinant Corynebacterium glutamicum strains. Notably, such a mannose fermentation process facilitated the synthesis of MOS, which has not been achieved under glucose fermentation and improved MG production by 2.6-fold than that using the same C-mole of glucose. This approach also allowed access to produce other kinds of mannosyl derivatives from starch.     

Metabolic engineering of Bacillus sp. for diacetyl production

Diacetyl, a highly valuable product that is extensively used as an ingredient of food, tobacco, and daily chemicals such as perfumes, can be produced from the nonenzymatic oxidative decarboxylation of α-acetolactate during bacterial fermentation and converted to acetoin and 2,3-butanediol by 2,3-butanediol dehydrogenase. In the present study, Bacillus sp. DL01, which gives high acetoin production, was metabolically engineered to improve diacetyl production. After the deletion of α-acetolactate decarboxylase (ALDC)-encoding gene (alsD) by homologous recombination, the engineered strain, named Bacillus sp. DL01-ΔalsD, lost ALDC activity and produced 1.53 g/L diacetyl without acetoin and 2,3-butanediol accumulation. The channeling of carbon flux into diacetyl biosynthetic pathway was amplified by an overexpressed α-acetolactate synthase (ALS)-encoding gene (alsS) in Bacillus sp. DL01-ΔalsD-alsS, which produced 4.02 g/L α-acetolactate and 1.94 g/L diacetyl, and the conversion from α-acetolactate to diacetyl was increased by 1-fold after 20 mM Fe³⁺ was added to the fermentation medium. A titer of 8.69 g/L diacetyl, the highest reported diacetyl production, was achieved by fed-batch fermentation in optimal conditions using the metabolically engineered strain of Bacillus sp. DL01-ΔalsD-alsS. These results are of great importance as a new method for the efficient production of diacetyl by food-safe bacteria.     

高产双乙酰乳球菌的研究进展

双乙酰是乳制品中一种重要的风味物质。大多数乳酸茵（如乳球菌）都可以发酵葡萄糖和柠檬酸产生双乙酰。描述了在乳球菌中从柠檬酸代谢和糖酵解途径来生成双乙酰的代谢途径,以及利用基因工程和代谢工程技术来提高乳球菌的双乙酰生成量的策略。     

Non-conventional hosts for the production of fuels and chemicals

Biotechnology offers a green alternative for the production of fuels and chemicals using microbes. Although traditional model hosts such as Escherichia coli and Saccharomyces cerevisiae have been widely studied and used, they may not be the best hosts for industrial application. In this review, we explore recent advances in the use of nonconventional hosts for the production of a variety of fuel, cosmetics, perfumes, food, and pharmaceuticals. Specifically, we highlight twenty-seven popular molecules with a special focus on recent progress and metabolic engineering strategies to enable improved production of fuels and chemicals. These examples demonstrate the promise of nonconventional host engineering.     

Reducing diacetyl production of wine by overexpressing <Emphasis Type="Italic">BDH1</Emphasis> and <Emphasis Type="Italic">BDH2</Emphasis> in <Emphasis Type="Italic">Saccharomyces uvarum</Emphasis>

As a byproduct of yeast valine metabolism during fermentation, diacetyl can produce a buttery aroma in wine. However, high diacetyl concentrations generate an aromatic off-flavor and poor quality in wine. 2,3-Butanediol dehydrogenase encoded by BDH1 can catalyze the two reactions of acetoin from diacetyl and 2,3-butanediol from acetoin. BDH2 is a gene adjacent to BDH1, and these genes are regulated reciprocally. In this study, BDH1 and BDH2 were overexpressed in Saccharomyces uvarum to reduce the diacetyl production of wine either individually or in combination. Compared with those in the host strain WY1, the diacetyl concentrations in the recombinant strains WY1-1 with overexpressed BDH1, WY1-2 with overexpressed BDH2 alone, and WY1-12 with co-overexpressed BDH1 and BDH2 were decreased by 39.87, 33.42, and 46.71%, respectively. BDH2 was only responsible for converting diacetyl into acetoin, but not for the metabolic pathway of acetoin to 2,3-butanediol in S. uvarum. This study provided valuable insights into diacetyl reduction in wine.     

Identification of metabolic engineering targets for the enhancement of 1,4-butanediol production in recombinant E. coli using large-scale kinetic models

Rational metabolic engineering methods are increasingly employed in designing the commercially viable processes for the production of chemicals relevant to pharmaceutical, biotechnology, and food and beverage industries. With the growing availability of omics data and of methodologies capable to integrate the available data into models, mathematical modeling and computational analysis are becoming important in designing recombinant cellular organisms and optimizing cell performance with respect to desired criteria. In this contribution, we used the computational framework ORACLE (Optimization and Risk Analysis of Complex Living Entities) to analyze the physiology of recombinant Escherichia coli producing 1,4-butanediol (BDO) and to identify potential strategies for improved production of BDO. The framework allowed us to integrate data across multiple levels and to construct a population of large-scale kinetic models despite the lack of available information about kinetic properties of every enzyme in the metabolic pathways. We analyzed these models and we found that the enzymes that primarily control the fluxes leading to BDO production are part of central glycolysis, the lower branch of tricarboxylic acid (TCA) cycle and the novel BDO production route. Interestingly, among the enzymes between the glucose uptake and the BDO pathway, the enzymes belonging to the lower branch of TCA cycle have been identified as the most important for improving BDO production and yield. We also quantified the effects of changes of the target enzymes on other intracellular states like energy charge, cofactor levels, redox state, cellular growth, and byproduct formation. Independent earlier experiments on this strain confirmed that the computationally obtained conclusions are consistent with the experimentally tested designs, and the findings of the present studies can provide guidance for future work on strain improvement. Overall, these studies demonstrate the potential and effectiveness of ORACLE for the accelerated design of microbial cell factories.     

Single cell mutant selection for metabolic engineering of actinomycetes

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g⁻¹ in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l⁻¹ in a second round of mutant selection. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.     

Automated engineering of synthetic metabolic pathways for efficient biomanufacturing

Metabolic engineering involves the engineering and optimization of processes from single-cell to fermentation in order to increase production of valuable chemicals for health, food, energy, materials and others. A systems approach to metabolic engineering has gained traction in recent years thanks to advances in strain engineering, leading to an accelerated scaling from rapid prototyping to industrial production. Metabolic engineering is nowadays on track towards a truly manufacturing technology, with reduced times from conception to production enabled by automated protocols for DNA assembly of metabolic pathways in engineered producer strains. In this review, we discuss how the success of the metabolic engineering pipeline often relies on retrobiosynthetic protocols able to identify promising production routes and dynamic regulation strategies through automated biodesign algorithms, which are subsequently assembled as embedded integrated genetic circuits in the host strain. Those approaches are orchestrated by an experimental design strategy that provides optimal scheduling planning of the DNA assembly, rapid prototyping and, ultimately, brings forward an accelerated Design-Build-Test-Learn cycle and the overall optimization of the biomanufacturing process. Achieving such a vision will address the increasingly compelling demand in our society for delivering valuable biomolecules in an affordable, inclusive and sustainable bioeconomy.     

Lactic acid bacteria as a cell factory: rerouting of carbon metabolism in Lactococcus lactis by metabolic engineering

Lactic acid bacteria display a relatively simple metabolism wherein the sugar is converted mainly to lactic acid. The extensive knowledge of metabolic pathways and the increasing information of the genes involved allows for the rerouting of natural metabolic pathways by genetic and physiological engineering. We discuss several examples of metabolic engineering of Lactococcus lactis for the production of important compounds, including diacetyl, alanine and exopolysaccharides.     

Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency

Precise prediction of prokaryotic translation efficiency can provide valuable information for optimizing bacterial host for the production of biochemical compounds or recombinant proteins. However, dynamic changes in mRNA folding throughout translation make it difficult to assess translation efficiency. Here, we systematically determined the universal folding regions that significantly affect the efficiency of translation in Escherichia coli. By assessing the specific regions for mRNA folding, we could construct a predictive design method, UTR Designer, and demonstrate that proper codon optimization around the 5′-proximal coding sequence is necessary to achieve a broad range of expression levels. Finally, we applied our method to control the threshold value of input signals switching on a genetic circuit. This should increase our understanding of the processes underlying gene expression and provide an efficient design principle for optimizing various biological systems, thereby facilitating future efforts in metabolic engineering and synthetic biology.     

Improvement of secondary metabolite production in Streptomyces by manipulating pathway regulation

Titer improvement is a constant requirement in the fermentation industry. The traditional method of “random mutation and screening” has been very effective despite the considerable amount of time and resources it demands. Rational metabolic engineering, with the use of recombinant DNA technology, provides a novel, alternative strategy for titer improvement that complements the empirical method used in industry. Manipulation of the specific regulatory systems that govern secondary metabolite production is an important aspect of metabolic engineering that can efficiently improve fermentation titers. In this review, we use examples from Streptomyces secondary metabolism, the most prolific source of clinically used drugs, to demonstrate the power and utility of exploiting natural regulatory networks, in particular pathway-specific regulators, for titer improvement. Efforts to improve the titers of fredericamycin, C-1027, platensimycin, and platencin in our lab are highlighted.     

Production of lactic acid from renewable materials by Rhizopus fungi

Lactic acid is a commonly occurring organic acid, which is valuable due to its wide use in food and food-related industries, and its potential for the production of biodegradable and biocompatible polylactate polymers. Lactic acid can be produced from renewable materials using various fungal species of the Rhizopus genus, which have advantages compared to the bacteria, including their amylolytic characteristics, low nutrient requirements and valuable fermentation by-product—fungal biomass. This paper reviews recent research in process engineering, metabolic and enzymetic mechanisms, and molecular biotechnology associated with lactic acid production by the Rhizopus fungi to get a better understanding of biochemical activities. The major process components: renewable materials, bioreactor systems and process modeling are reviewed. The role of key bioprocess parameters, such as nutrient composition, pH and growth morphology, involved in the production of lactic acid is discussed in detail. In addition, recent advances in simultaneous saccharification and fermentation, molecular genetic approaches, and enzymetic and metabolic pathways involved in the production of lactic acid by fungal strains are discussed.     

Systems metabolic engineering of Corynebacterium glutamicum for the efficient production of β-alanine

β-Alanine is an important β-amino acid with a growing demand in a wide range of applications in chemical and food industries. However, current industrial production of β-alanine relies on chemical synthesis, which usually involves harmful raw materials and harsh production conditions. Thus, there has been increasing demand for more sustainable, yet efficient production process of β-alanine. In this study, we constructed Corynebacterium glutamicum strains for the highly efficient production of β-alanine through systems metabolic engineering. First, aspartate 1-decarboxylases (ADCs) from seven different bacteria were screened, and the Bacillus subtilis ADC showing the most efficient β-alanine biosynthesis was used to construct a β-alanine-producing base strain. Next, genome-scale metabolic simulations were conducted to optimize multiple metabolic pathways in the base strain, including phosphotransferase system (PTS)-independent glucose uptake system and the biosynthesis of key precursors, including oxaloacetate and L-aspartate. TCA cycle was further engineered for the streamlined supply of key precursors. Finally, a putative β-alanine exporter was newly identified, and its overexpression further improved the β-alanine production. Fed-batch fermentation of the final engineered strain BAL10 (pBA2_tr18) produced 166.6 g/L of β-alanine with the yield and productivity of 0.28 g/g glucose and 1.74 g/L/h, respectively. To our knowledge, this production performance corresponds to the highest titer, yield and productivity reported to date for the microbial fermentation.     

Lactococcus lactis as a Cell Factory for High-Level Diacetyl Production

We report the engineering of Lactococcus lactis for the efficient conversion of sugar into diacetyl by combining NADH-oxidase overproduction and α-acetolactate decarboxylase inactivation. Eighty percent of the carbon flux was found to be rerouted via α-acetolactate to the production of diacetyl by preloading the cells with NADH-oxidase before their use as a cell factory.