Key Process Conditions for Production of C4 Dicarboxylic Acids in Bioreactor Batch Cultures of an Engineered Saccharomyces cerevisiae Strain

A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter⁻¹ of malate at a yield of 0.42 mol (mol glucose)⁻¹ in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO₃, was required for efficient C₄ dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO₃ dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate titers further increased, reaching 0.29 mol (mol glucose)⁻¹, whereas malate formation strongly decreased. Although fully aerobic conditions could be achieved, it was found that moderate oxygen limitation benefitted malate production. In conclusion, malic acid production with the engineered S. cerevisiae strain could be successfully transferred from shake flasks to 1-liter batch bioreactors by simultaneous optimization of four process parameters (pH and concentrations of CO₂, calcium, and O₂). Under optimized conditions, a malate yield of 0.48 ± 0.01 mol (mol glucose)⁻¹ was obtained in bioreactors, a 19% increase over yields in shake flask experiments.In recent years, biologically produced 1,4-dicarboxylic acids (succinate, malate, and fumarate) have attracted great interest as more sustainable replacements for oil-derived commodity chemicals, such as maleic anhydride (50). Malate is currently mainly produced via petrochemical routes for use in food and beverages (18). Development of a biotechnological production process started in the early 1960s with the investigation of the natural malate producer Aspergillus flavus (2). Although process improvements eventually resulted in high product yields and productivities (6), the potential production of aflatoxins (20) prevented the use of this filamentous fungus in industry. Other investigated natural malate-producing fungi (listed in reference 51) produced insufficient malate for industrial use. With the rational design options of metabolic engineering, microorganisms that do not naturally produce large amounts of malic acid may also be considered as production platforms. Wild-type Saccharomyces cerevisiae strains produce little if any malate but would be an interesting starting point for the construction of an efficient malate producer. This yeast has a relatively high tolerance to organic acids and low pH, and due to its role as a model organism in research, a well-developed metabolic engineering toolbox is available. In addition, wild-type S. cerevisiae strains have GRAS (Generally Regarded As Safe) status, so that modified strains are more likely to be allowed in the production of food-grade malic acid.One of the main challenges in the development of an organic acid-producing strain of S. cerevisiae has been the elimination of ethanol formation, which in wild-type strains occurs even under aerobic conditions when glucose concentrations are high (45). Deletion of the pyruvate decarboxylase-encoding genes was found to prevent ethanolic fermentation (17). After evolutionary engineering to remove the growth defects usually associated with pyruvate decarboxylase-negative S. cerevisiae strains, a strain was obtained that produced large amounts of pyruvate, a direct precursor to malate, when grown on glucose (42). Subsequent overexpression of the anaplerotic enzyme pyruvate carboxylase, a cytosolically relocalized malate dehydrogenase and a heterologous malate transporter from Schizosaccharomyces pombe led to a strain that produced significant amounts of malate (51). Cultivation in calcium carbonate (CaCO₃)-buffered shake flasks resulted in malate titers of up to 59 g liter⁻¹ at a yield of 0.42 mol (mol glucose)⁻¹.There are many differences between cultivation in shake flasks and cultivation in (laboratory or industrial) bioreactors. As shake flask cultures lack online pH monitoring and control, there is often significant pH variation over time. The pH is of particular importance. If the yeast can be persuaded to produce organic acids at lower pH values, this reduces the need for active neutralization and thereby reduces by-product formation such as gypsum. However, thermodynamic constraints on acid export, as well as increased stress levels from (undissociated) acid and the low pH, often limit the ability of the microorganisms to produce acids at low pH (32, 43). For this reason, the poorly soluble compound CaCO₃ has traditionally been used to maintain a near-neutral pH in malic acid-producing microbial cultures (6, 29, 51). Adding CaCO₃ also gives increased concentrations of bicarbonate (and thereby CO₂), a substrate for pyruvate carboxylase in the carboxylation of pyruvate (a C₃ carbon molecule) to oxaloacetate (C₄ carbon), as well as calcium. Calcium is known to be involved in cellular signaling pathways (22, 26, 33, 46) and to influence pyruvate carboxylase activity (21, 24). Finally, oxygen transfer rates in shake flasks are often poor compared to those in stirred (laboratory) bioreactors. The formation of significant concentrations (25 g liter⁻¹) of glycerol, a well-known redox sink in S. cerevisiae (41), in shake flask cultures of the engineered malate-producing strain (51) was a strong indication of oxygen limitation.Initial experiments in aerobic, pH-controlled bioreactor cultures of the malate- and succinate-producing Saccharomyces cerevisiae strain RWB525 yielded only low concentrations of these C₄ dicarboxylic acids. The goal of the present study was to identify process parameters that explain the different production levels in shake flask and bioreactor cultures. To this end, we analyzed, both separately and in combination, the impact of culture pH and concentrations of calcium, carbon dioxide, and oxygen on the production of malate and succinate.