Improvement of D‐lactic acid production at low pH through expressing acid‐resistant gene IoGAS1 in engineered Saccharomyces cerevisiae

Abstract

BACKGROUND Blending d‐lactic acid (d‐LA) with l‐lactic acid can significantly improve the thermostability of polylactic (PLA). Although microbial production of d‐LA under acidic conditions is beneficial for the reduction of production costs, the yield is low due to the acidic toxicity of the source strains. Herein, an Issatchenkia orientalis glycosylphosphatidylinositol‐anchored protein IoGas1, which is required for resistance to low pH and salt stress, was expressed in the YIP‐J‐C‐D‐A1 yeast strain. This strain was integrated with Escherichia coli d‐lactate dehydrogenase gene and several attenuated key pathway genes, including pyruvate decarboxylases (PDC1, PDC6), JEN1 (a monocarboxylate transporter), d‐lactate dehydrogenase1 (DLD1), l‐lactate cytochrome‐c oxidoreductase (CYB2) and alcohol dehydrogenase 1(ADH1). RESULTS The results revealed that the production of d‐LA by the modified strain YIP‐I‐J‐C‐D‐A1 was remarkably improved and reached 85.3 g L–1 d‐LA, with a yield of 0.71 g g–1 and a productivity of 1.20 g L h–1 in batch‐fed fermentation. The d‐LA production of the YIP‐I‐J‐C‐D‐A1 strain (CGMCC2.5785) was further improved by attenuating the ethanol and glycerol pathways. The resulting strain YIP‐A15G12 (CGMCC2.5803) produced 92.0 g L–1 d‐LA with a yield of 0.70 g g–1 and a productivity of 1.21 g L h–1 in batch‐fed fermentation at a final pH of 3.58. CONCLUSION Taken together, the expression of the acid‐resistant gene IoGAS1 in a modified yeast strain can significantly improve the efficiency of producing d‐LA at low pH, which may prove beneficial for the industrial production of the biodegradable material, PLA.

Glucose assimilation rate determines the partition of flux at pyruvate between lactic acid and ethanol in Saccharomyces cerevisiae

“…For example, the I. orientalis GPI‐anchored protein encoded by IoGAS1 confers low pH tolerance when expressed in engineered S. cerevisiae strains. [ 13,43 ]…”

“…For example, the I. orientalis GPI-anchored protein encoded by IoGAS1 confers low pH tolerance when expressed in engineered S. cerevisiae strains. [13,43] Future researchers aiming to create lactic acid and other pyruvate-derived molecules in S. cerevisiae strains expressing native PDC enzymes may find some inspiration from the physio-logical changes present during xylose metabolism. Using xylose as a carbon source has been shown to increase yields of a variety of products, such as isobutanol, [44,45] 2,3-butanediol, [46,47] poly-3-Dhydroxybutyrate, [48,49] squalene, and amorphadiene.…”

“…[11] Because of this, production of large amounts of lactic acid from glucose has generally required deletion of pyruvate decarboxylase (PDC) enzymes encoded by the PDC1, PDC5, and PDC6 genes. [12][13][14] However, this deletion of PDCs comes with critical drawbacks such as C 2 auxotrophy which requires supplementation with either acetate or ethanol, and reduced growth rate and fermentation speed. As such, it is desirable to engineer a yeast strain capable of efficient production of non-ethanol compounds derived from pyruvate, such as lactic acid, without deleting PDC enzymes.…”

The cell wall and the response and tolerance to stresses of biotechnological relevance in yeasts

“…Enhanced tolerance to both acetic and formic acids at low pH (pH 2.4 or below) by expressing the GAS1 gene of Issatchenkia orientalis in S. cerevisiae was reported ( Wada et al, 2020 ). Also, the overexpression of GAS1 in S. cerevisiae led to increased lactic acid productivity at low pH ( Zhong et al, 2021 ), reinforcing the importance of GAS1 , encoding a beta-1,3-glucanosyltransferase, in this context ( Baek et al, 2017 ).…”

Just around the Corner: Advances in the Optimization of Yeasts and Filamentous Fungi for Lactic Acid Production

“…Again, Peetermans et al [ 29 ] reviewed other major aspects of lactic acid tolerance in S. cerevisiae , which include amino acid metabolism and cell wall composition. It is worth noting that gains in lactic acid production have been obtained from the introduction of genes that confer general acid tolerance, but these tend to be marginal [ 73 , 76 ], as the toxicity of small organic acids does not derive merely from lowering the pH.…”