Optimization of D-lactic acid biosynthesis from diverse carbon sources in mutant Lactobacillus delbrueckii subsp. bulgaricus via random mutagenesis

This study aims to augment the D-lactic acid biosynthetic capacity of Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 through random mutagenesis. The mutant strain, Mut_N23, developed through synergistic application of ultraviolet (UV) irradiation and chemical mutagenesis using N-methyl-N′-nitro-N-nitrosoguanidine (NTG), exhibited 97% increase in D-lactic acid production and 37% enhancement in glucose uptake rate at flask level. Mut_N23 consistently produced optically pure D-lactic acid across seven generations, efficiently metabolizing lactose and sucrose to yield 4.47 g L^− 1 and 3.38 g L^− 1 of D-lactic acid, respectively. Optimal conditions identified through One-Factor-At-a-Time (OFAT), and Response Surface Methodology (RSM) facilitated maximum D-lactic acid concentration of 7.88 g L^− 1 (300% increase) from lactose-MRS (deMan Rogosa Sharpe) with specific productivity of 0.110 g g^− 1 h^− 1. When lactose was replaced with whey permeate as an application, 4.89 g L^− 1 (140% increase) of D-lactic acid was obtained, with specific productivity of 0.066 g g^− 1 h^− 1 in lab-scale bioreactor setups, achieving 99.09% optical purity. Transcriptomics and enzymatic activity analyses substantiated enhanced performance of Mut_N23 signifying beneficial random mutations. Furthermore, characterization of purified D-lactic acid derived from whey permeate using Fourier Transform Infrared (FTIR) spectroscopy and proton Nuclear Magnetic Resonance (NMR) spectroscopy demonstrated parity with commercially available standards. This study highlights Mut_N23’s potential for efficient D-lactic acid production exploiting a spectrum of carbon sources, providing a foundation for future metabolic engineering to enhance biosynthetic productivity.